CN108624697B - A kind of 21 gene detecting kit of breast cancer and its detection method - Google Patents

A kind of 21 gene detecting kit of breast cancer and its detection method Download PDF

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CN108624697B
CN108624697B CN201810999037.8A CN201810999037A CN108624697B CN 108624697 B CN108624697 B CN 108624697B CN 201810999037 A CN201810999037 A CN 201810999037A CN 108624697 B CN108624697 B CN 108624697B
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CN108624697A (en
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唐荣
苏丽
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Wo Wo Medical Technology (shanghai) Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development

Abstract

The invention discloses a kind of 21 gene detecting kit of breast cancer and its detection methods；The kit includes the primer and probe for detecting ACTB, GAPDH, RPLPO, GUS, TFRC, CD68, BAG1, GSTM1, HER2, GRB7, MMP11, CTSL2, KI67, STK15, CCNB1, MYBL2, Survivin, ESR1, PGR, BCL2 and SCUBE2 gene respectively, 21 genes respectively devise two pairs of primers and 2 specific probes in kit, the primer and probe mixed liquor of each gene is placed in a reaction tube, same buffer and program are used when expanding on QPCR, it is easy to operate, save detection time and obtain clear and accurate result；This kit is widely applicable, and scientific research and clinical detection are all suitable for, single or multiple sample, can use this kit, testing result is quick and precisely.

Description

A kind of 21 gene detecting kit of breast cancer and its detection method
Technical field
The present invention relates to gene technology fields, and in particular to a kind of 21 gene detecting kit of breast cancer and its detection side Method.
Background technique
Breast cancer is one of the most common malignant tumors in women, in the past 20 years, by operation centered on, chemotherapy, radiotherapy, Endocrine therapy is that the multidisciplinary synthesis treatment mode of auxiliary obtains significant progress, keeps the recurrence of breast cancer and the death rate significant It reduces, but still lack effective method can relatively accurately predict the risk of recurrence of patient and give corresponding treatment.
The recurring risk assessment system of breast cancer is to detect tumor tissues by accurately calculating based on the method for RT-QPCR In 16 destination gene expressions relevant to tumour, i.e. 21 genetic test of breast cancer obtains cream by gene expression amount calculating The recurrence of gland cancer is scored (RS), by RS value assess 10 years breast cancer relapses of therapeutic effect and prediction of individuation risk and Receive the benefit ratio of chemotherapy.21 genetic test of breast cancer is American Society of Clinical Oncology (ASCO) and the comprehensive cancer net of US National The multi-gene expression check and evaluation and service for Prognosis in Breast Cancer and chemotherapy of network (NCCN) combine recommendation, can be to avoid resource Waste and the over-treatment to the low danger person in part.21 gene of breast cancer includes that 16 breast cancer related genes and 5 refer to base Cause, i.e. increment group (Ki67, STK15, Survivin, CCNB1 and MYBL2), invasion group (MMP1 1 and CTSL2), HER-2 Group (GRB7 and HER2), estrogen group (ER, PgR, BCL2 and SCUBE2), other genomes (GSTM1, CD68 and BAG1), Reference gene group (ACTB, GAPDH, RPLP0, Gus and TFRc).
21 genetic tests needs extract RNA from specimens paraffin embedding slices, and the RNA extracted from the sample is inevitably The case where degrading.So we need to solve still ask what 21 genes were detected in RNA degradation Topic.The kit of current 21 genetic test generally uses SYBR Green method, Taqman fluorescent PCR method.SYBR Green method is only Pair of primers need to be designed, needs to be added I fluorescent dye of SYBR Green in reaction, the shortcomings that this method is PCR Poor specificity, sensitivity is not high, and result is not very accurate.Taqman fluorescent PCR method, needs design primer and probe, Simple Taqman method need to only design pair of primers and a probe, and multiple fluorescence PCR need to design multipair primer and Probe, and to guarantee not interfere with each other between each primer of multiplex PCR and each probe between do not interfere with each other, this is just Requirement to primer and probe is relatively high, and design difficulty is big；In addition, multiplex PCR has one to the buffer and QPCR instrument of PCR Fixed requirement.21 genetic tests have 21 genes to need to detect, and 21 genes are placed in same PCR pipe and are detected not now It is real, so existing multiple fluorescence PCR method is 21 genes to be divided into eight groups to detect, that is, to add in each reaction tube 2-3 gene, this adds increased the complexity of experimental implementation, and obtained experimental data is also not very clear, and And we do not know whether several genes in the same pipe have interference between each other yet.It would therefore be desirable to find a kind of spy Anisotropic, high sensitivity, easy to operate, accurate 21 gene detecting kit of data and method.
Summary of the invention
It is an object of the invention in order to overcome specificity sensitivity existing in the prior art not good enough or complicated for operation Defect provides a kind of 21 gene detecting kit of breast cancer and its detection method.
The purpose of the present invention is achieved through the following technical solutions:
In a first aspect, the present invention relates to a kind of 21 gene detecting kit of breast cancer, it is characterized in that 21 genes are as follows: ACTB、GAPDH、RPLPO、GUS、TFRC、CD68、BAG1、GSTM1、HER2、GRB7、MMP11、CTSL2、KI67、STK15、 CCNB1, MYBL2, Survivin, ESR1, PGR, BCL2 and SCUBE2, primer sequence and TaqMan corresponding to 21 genes are visited Needle sequence difference is as follows:
21 genes respectively devise two pairs of primers and 2 specific probes, primer corresponding to 21 genes in kit Sequence and TaqMan probe sequence difference are as follows:
(1) detect ACTB gene primer sequence are as follows: SEQ ID NO.1 and SEQ ID NO.2 and SEQ ID NO.3 and SEQ ID NO.4 detects the TaqMan probe sequence of ACTB gene are as follows: SEQ ID NO.5 and SEQ ID NO.6；Particular sequence It is as follows:
ACTB:
F1:5'-CCAACTTGAGATGTATGAAG -3'(SEQ ID NO.1)
R1:5'-GTCAGTGTACAGGTAAGC -3'(SEQ ID NO.2)
F2:5'-TGGAGAAGAGCTACGA -3'(SEQ ID NO.3)
R2:5'-GAAGGAAGGCTGGAAG -3'(SEQ ID NO.4)
P1:5'FAM-CTGCCTCCACCCACTCCC-3'MGB (SEQ ID NO.5)
P2:5'VIC-CGGAACCGCTCATTGCCA-3'MGB (SEQ ID NO.6)；
(2) detect GAPDH gene primer sequence are as follows: SEQ ID NO.7 and SEQ ID NO.8 and SEQ ID NO.9 and SEQ ID NO.10 detects the TaqMan probe sequence of GAPDH gene are as follows: SEQ ID NO.11 and SEQ ID NO.12；Specifically Sequence is as follows:
GAPDH:
F1:5'-GCCTCCAAGGAGTAAGA -3'(SEQ ID NO.7)
R1:5'-GCAGTGAGGGTCTCTC -3'(SEQ ID NO.8)
F2:5'-CCATGACAACTTTGGTATCA -3'(SEQ ID NO.9)
R2:5'-GCCATCCACAGTCTTCA -3'(SEQ ID NO.10)
P1:5'FAM-TCCTCTTGTGCTCTTGCTGG-3'MGB (SEQ ID NO.11)
P2:5'VIC-AGTCCATGCCATCACTGCCA-3'MGB (SEQ ID NO.12)；
(3) primer sequence of RPLPO gene is detected are as follows: SEQ ID NO.13 and SEQ ID NO.14 and SEQ ID NO.15 With SEQ ID NO.16, the TaqMan probe sequence of RPLPO gene is detected are as follows: SEQ ID NO.17 and SEQ ID NO.18；Tool Body sequence is as follows:
RPLPO:
F1:5'--GGTTGAAGCCAAGGAAGA -3'(SEQ ID NO.13)
R1:5'-TGGTGATTAGTCAAAGAGACC -3'(SEQ ID NO.14)
F2:5'-GGGCACCATTGAAATCCA -3'(SEQ ID NO.15)
R2:5'-GGAGATGTTGAGCATGTTCA -3'(SEQ ID NO.16)
P1:5'FAM-ATCCTCGTCCGACTCCTCCG-3'MGB (SEQ ID NO.17)
P2:5'VIC-CGTGGCTTCGCTGGCTCC-3'MGB (SEQ ID NO.18)；
(4) detect gus gene primer sequence are as follows: SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22 detects the TaqMan probe sequence of gus gene are as follows: SEQ ID NO.23 and SEQ ID NO.24；Specific sequence It arranges as follows:
GUS:
F1:5'-CCACAGCAGCAGAACA-3'(SEQ ID NO.19)
R1:5'- CTGCTAGAATAGATGACCACAA-3'(SEQ ID NO.20)
F2:5'-GGTGTCAACAAGCATGAGA -3'(SEQ ID NO.21)
R2:5'-CAGCGAAGCAGGTTGAA -3'(SEQ ID NO.22)
P1:5'FAM-CCTCCTGGACTGTTCACGGC-3'MGB (SEQ ID NO.23)
P2:5'VIC-TCCTTCACCAGCAGCGGC-3'MGB (SEQ ID NO.24)；
(5) primer sequence of TFRC gene is detected are as follows: SEQ ID NO.25 and SEQ ID NO.26 and SEQ ID NO.27 With SEQ ID NO.28, the TaqMan probe sequence of TFRC gene is detected are as follows: SEQ ID NO.29 and SEQ ID NO.30；Specifically Sequence is as follows:
TFRC:
F1:5'-GGACTGACATTTAGCGTAG -3'(SEQ ID NO.25)
R1:5'-GCCACATGCTTTCATTTAAG -3'(SEQ ID NO.26)
F2:5'-GAGAGGATTCCTGAGTTGAA -3'(SEQ ID NO.27)
R2:5'-TCCAGGTTCAATTCAACATCA -3'(SEQ ID NO.28)
P1:5'FAM-TGCGTAACACCCGAACCAGG-3'MGB (SEQ ID NO.29)
P2:5'VIC-CACGAACTGACCAGCGACCT-3'MGB (SEQ ID NO.30)；
(6) detect CD68 gene primer sequence be SEQ ID NO.31 and SEQ ID NO.32 and SEQ ID NO.33 and SEQ ID NO.34 detects the TaqMan probe sequence of CD68 gene are as follows: SEQ ID NO.35 and SEQ ID NO.36；Specific sequence It arranges as follows:
CD68:
F1:5'-GGCAGAACTGCTTGAA -3'(SEQ ID NO.31)
R1:5'-GCGATCTTGGCTTAGTG -3'(SEQ ID NO.32)
F2:5'-ACCTGCTTCTCTCATTCCA -3'(SEQ ID NO.33)
R2:5'-CTCCACCGCCATGTAGA -3'(SEQ ID NO.34)
P1:5'FAM-CGGCTCACTGCAACCTCCA-3'MGB (SEQ ID NO.35)
P2:5'VIC-CCTTCTGCTGGAGGTCCTGC-3'MGB (SEQ ID NO.36)；
(7) primer sequence of BAG1 gene is detected are as follows: SEQ ID NO.37 and SEQ ID NO.38 and SEQ ID NO.39 With SEQ ID NO.40, the TaqMan probe sequence of BAG1 gene is detected are as follows: SEQ ID NO.41 and SEQ ID NO.42；Specifically Sequence is as follows:
BAG1:
F1:5'-GCTACTACCTCTGCTTGA -3'(SEQ ID NO.37)
R1:5'-GTGAGTTGAATTCTGAAGGA -3'(SEQ ID NO.38)
F2:5'-CCACAGGCCATGAGATAA -3'(SEQ ID NO.39)
R2:5'-GGTTCTAACCTTCTGAAGACA -3'(SEQ ID NO.40)
P1:5'FAM-TCCTCACCTTCCTTCCTCCA-3'MGB (SEQ ID NO.41)
P2:5'VIC-CCGTTCCTTGGATGGAGCCT-3'MGB (SEQ ID NO.42)；
(8) primer sequence of GSTM1 gene is detected are as follows: SEQ ID NO.43 and SEQ ID NO.44 and SEQ ID NO.45 With SEQ ID NO.46, the TaqMan probe sequence of GSTM1 gene is detected are as follows: SEQ ID NO.47 and SEQ ID NO.48；Tool Body sequence is as follows:
GSTM1:
F1:5'-CCCATCATTACCCTTCC -3'(SEQ ID NO.43)
R1:5'-GCCCTTTAAAGCAGACA -3'(SEQ ID NO.44)
F2:5'-GAGGGCTTGGAGAAGAA -3'(SEQ ID NO.45)
R2:5'-GACAGCCATCTTTGAGAAA -3'(SEQ ID NO.46)
P1:5'FAM-AGCCAGCCTGACCTTCCTTC-3'MGB (SEQ ID NO.47)
P2:5'VIC-AGTCCAGCCGCTTCCTCC-3'MGB (SEQ ID NO.48)；
(9) primer sequence of HER2 gene is detected are as follows: SEQ ID NO.49 and SEQ ID NO.50 and SEQ ID NO.51 With SEQ ID NO.52, the TaqMan probe sequence of HER2 gene is detected are as follows: SEQ ID NO.53 and SEQ ID NO.54；Specifically Sequence is as follows:
HER2:
F1:5'-CTCCGACCACTTCCAG -3'(SEQ ID NO.49)
R1:5'-CCCTTCCAGCCATCTG -3'(SEQ ID NO.50)
F2:5'-GGTGGTTGGCATTCTGA -3'(SEQ ID NO.51)
R2:5'-CCGCATCGTGTACTTC -3'(SEQ ID NO.52)
P1:5'FAM-CTGCCATGCCAGGAACCTGT-3'MGB (SEQ ID NO.53)
P2:5'VIC-TTCTGCTGCCGTCGCTTGA-3'MGB (SEQ ID NO.54)；
(10) primer sequence of GRB7 gene is detected are as follows: SEQ ID NO.55 and SEQ ID NO.56 and SEQ ID NO.57 With SEQ ID NO.58, the TaqMan probe sequence of GRB7 gene is detected are as follows: SEQ ID NO.59 and SEQ ID NO.60；Specifically Sequence is as follows:
GRB7:
F1:5'-CCTCCCTCAGATAGAA -3'(SEQ ID NO.55)
R1:5'-GCTAGGAGAAGGAGAG -3'(SEQ ID NO.56)
F2:5'-GGCTTCGGATCTTCTGA -3'(SEQ ID NO.57)
R2:5'-CCGTACTTGAAGAGGC -3'(SEQ ID NO.58)
P1:5'FAM-CCACTCCAGTCCACTCCTGA-3'MGB (SEQ ID NO.59)
P2:5'VIC-AGATGAGCAGAGCCGCACC-3'MGB (SEQ ID NO.60)；
(11) primer sequence of MMP11 gene is detected are as follows: SEQ ID NO.61 and SEQ ID NO.62 and SEQ ID NO.63 and SEQ ID NO.64 detects the TaqMan probe sequence of MMP11 gene are as follows: SEQ ID NO.65 and SEQ ID NO.66；Particular sequence is as follows:
MMP11:
F1:5'-AGGTCAGGTCTTGGTA -3'(SEQ ID NO.61)
R1:5'-CTGGATTCTGGAGAACA -3'(SEQ ID NO.62)
F2:5'--CCAGATGCCTGTGAGGA -3'(SEQ ID NO.63)
R2:5'-AGCCCGCTTTGAAGAA -3'(SEQ ID NO.64)
P1:5'FAM-TTCTGGCTGACAATCCTGGA-3'MGB (SEQ ID NO.65)
P2:5'VIC-TCCACCATCCGAGGCGAGC-3'MGB (SEQ ID NO.66)；
(12) primer sequence of CTSL2 gene is detected are as follows: SEQ ID NO.67 and SEQ ID NO.68 and SEQ ID NO.69 and SEQ ID NO.70；Detect the TaqMan probe sequence of CTSL2 gene are as follows: SEQ ID NO.71 and SEQ ID NO.72；Particular sequence is as follows:
CTSL2:
F1:5'-CCTAGATGGTATAGCCTATTAC -3'(SEQ ID NO.67)
R1:5'-AGCACAGTGATGTGTTG -3'(SEQ ID NO.68)
F2:5'-GGCTGGTCTTGAACTCA -3'(SEQ ID NO.69)
R2:5'-TGGCTCACATCTGTAATC -3'(SEQ ID NO.70)
P1:5'FAM-TGGTACAGCCTGTTGCTCCT-3'MGB (SEQ ID NO.71)
P2:5'VIC-CCACCTGCCTCAGCCTCC-3'MGB (SEQ ID NO.72)；
(13) primer sequence of KI67 gene is detected are as follows: SEQ ID NO.73 and SEQ ID NO.74 and SEQ ID NO.75 With SEQ ID NO.76, the TaqMan probe sequence of KI67 gene is detected are as follows: SEQ ID NO.77 and SEQ ID NO.78；Specifically Sequence is as follows:
KI67:
F1:5'-CTCTGTACCTACCATCTCC -3'(SEQ ID NO.73)
R1:5'-CAGGAGGGAAAGTTCCA -3'(SEQ ID NO.74)
F2:5'-GGGAAAGTAGGTGTGAAAGA -3'(SEQ ID NO.75)
R2:5'-CCATCTCCTGCTGTCTC -3'(SEQ ID NO.76)
P1:5'FAM-AGGCACCTCCAACCACCACA-3'MGB (SEQ ID NO.77)
P2:5'VIC-ACCAGTCGGCAAGCTCACAC-3'MGB (SEQ ID NO.78)；
(14) primer sequence of STK15 gene is detected are as follows: SEQ ID NO.79 and SEQ ID NO.80 and SEQ ID NO.81 and SEQ ID NO.82 detects the TaqMan probe sequence of STK15 gene are as follows: SEQ ID NO.83 and SEQ ID NO.84；Particular sequence is as follows:
STK15:
F1:5'-ACAGGAGGCAAATCC -3'(SEQ ID NO.79)
R1:5'-CAGGTACTAGGAAGGTTATTG -3'(SEQ ID NO.80)
F2:5'-GGGTGGTCAGTACATGA -3'(SEQ ID NO.81)
R2:5'-CATCCGACCTTCAATCA -3'(SEQ ID NO.82)
P1:5'FAM-TGACCACTCTGCCCTGACCC-3'MGB (SEQ ID NO.83)
P2:5'VIC-TCCATCTTCCAGGAGGACCA-3'MGB (SEQ ID NO.84)；
(15) primer sequence of CCNB1 gene is detected are as follows: SEQ ID NO.85 and SEQ ID NO.86 and SEQ ID NO.87 and SEQ ID NO.88 detects the TaqMan probe sequence of CCNB1 gene are as follows: SEQ ID NO.89 and SEQ ID NO.90；Particular sequence is as follows:
CCNB1:
F1:5'-CTGTCAAGAACAAGTATGC -3'(SEQ ID NO.85)
R1:5'-CACAGCCTTGGCTAAA -3'(SEQ ID NO.86)
F2:5'-ATCGGTTCATGCAGAATAATTGA -3'(SEQ ID NO.87)
R2:5'-GGAGGGTACATTTCTTCATATTTG -3'(SEQ ID NO.88)
P1:5'FAM-AAGATCAGCACTCTACCACAGC-3'MGB (SEQ ID NO.89)
P2:5'VIC-TGGCAGTGACACCAACCAGC-3'MGB (SEQ ID NO.90)；
(16) primer sequence of MYBL2 gene is detected are as follows: SEQ ID NO.91 and SEQ ID NO.92 and SEQ ID NO.93 and SEQ ID NO.94 detects the TaqMan probe sequence of MYBL2 gene are as follows: SEQ ID NO.95 and SEQ ID NO.96；Particular sequence is as follows:
MYBL2:
F1:5'-CAGACTCTCAGGTGGA -3'(SEQ ID NO.91)
R1:5'-GACCTGGAAGTGGAAC -3'(SEQ ID NO.92)
F2:5'-GCCCTCTGTGCTCAAGA -3'(SEQ ID NO.93)
R2:5'-CAGACTGGTGCTATTCTCA -3'(SEQ ID NO.94)
P1:5'FAM-AGCACCAGGAGCCGCC-3'MGB (SEQ ID NO.95)
P2:5'VIC-CACACGCCTCTTCCTCTGCC-3'MGB (SEQ ID NO.96)；
(17) primer sequence of Survivin gene is detected are as follows: SEQ ID NO.97 and SEQ ID NO.98 and SEQ ID NO.99 and SEQ ID NO.100, the TaqMan probe sequence of detection Survivin gene are SEQ ID NO.101 and SEQ ID NO.102；Particular sequence is as follows:
Survivin:
F1:5'-GCCATCCTTAAAACCAGA -3'(SEQ ID NO.97)
R1:5'-CGGTTGCACTTAGACAA -3'(SEQ ID NO.98)
F2:5'-CCTTCACATCTGTCACGA -3'(SEQ ID NO.99)
R2:5'-GCTGCCTCCAAAGAAAG -3'(SEQ ID NO.100)
P1:5'FAM-CCTCATGGCTACCAGCACCT-3'MGB (SEQ ID NO.101)
P2:5'VIC-ACGCAGTCCGCCCAGGT-3'MGB (SEQ ID NO.102)；
(18) primer sequence of ESR1 gene is detected are as follows: SEQ ID NO.103 and SEQ ID NO.104 and SEQ ID NO.105 and SEQ ID NO.106 detects the TaqMan probe sequence of ESR1 gene are as follows: SEQ ID NO.107 and SEQ ID NO.108；Particular sequence is as follows:
ESR1:
F1:5'-GCACCCTAGAAACAACATA -3'(SEQ ID NO.103)
R1:5'-GTCTCTATAACCAATGACCTC -3'(SEQ ID NO.104)
F2:5'-CTGGTGATTGTCAATTCATTCA -3'(SEQ ID NO.105)
R2:5'-GCAGTGTATCAAGTTAAAATAGTC -3'(SEQ ID NO.106)
P1:5'FAM-AGGTGCCTGAGACACAGACC-3'MGB (SEQ ID NO.107)
P2:5'VIC-TCTGCCTTCCCTGTGTGCCC-3'MGB (SEQ ID NO.108)；
(19) primer sequence of PGR gene is detected are as follows: SEQ ID NO.109 and SEQ ID NO.110 and SEQ ID NO.111 and SEQ ID NO.112 detects the TaqMan probe sequence of PGR gene are as follows: SEQ ID NO.113 and SEQ ID NO.114；Particular sequence is as follows:
PGR:
F1:5'-TGCCAAGAAGGTGAAAC -3'(SEQ ID NO.109)
R1:5'-ACAGCTTTGCATTGTCA -3'(SEQ ID NO.110)
F2:5'-CATGGAATGTCAACCTGTAA -3'(SEQ ID NO.111)
R2:5'-GCACAGTCCTATGTCAG -3'(SEQ ID NO.112)
P1:5'FAM-CCATCACTGCCAGGGACCCA-3'MGB (SEQ ID NO.113)
P2:5'VIC-CTCCACAGTGCCCATCATCA-3'MGB (SEQ ID NO.114)；
(20) primer sequence of BCL2 gene is detected are as follows: SEQ ID NO.115 and SEQ ID NO.116 and SEQ ID NO.117 and SEQ ID NO.118 detects the TaqMan probe sequence of BCL2 gene are as follows: SEQ ID NO.119 and SEQ ID NO.120；Particular sequence is as follows:
BCL2:
F1:5'-GGGTATGAAGGACCTGTA -3'(SEQ ID NO.115)
R1:5'- GTCGTACCACAAACTTCAAA-3'(SEQ ID NO.116)
F2:5'-GTGAGGAGAGGCAATGA -3'(SEQ ID NO.117)
R2:5'-CCTTGCTTTAAACTCACAG -3'(SEQ ID NO.118)
P1:5'FAM-TGCCTCTGCGAAGAACCTTG-3'MGB (SEQ ID NO.119)
P2:5'VIC-CAGCCAACGTGCCATGTGC-3'MGB (SEQ ID NO.120)；
(21) primer sequence of SCUBE2 gene is detected are as follows: SEQ ID NO.121 and SEQ ID NO.122 and SEQ ID NO.123 and SEQ ID NO.124 detects the TaqMan probe sequence of SCUBE2 gene are as follows: SEQ ID NO.125 and SEQ ID NO.126；Particular sequence is as follows:
SCUBE2:
F1:5'-CCCAGTCTCAAATGTGA -3'(SEQ ID NO.121)
R1:5'-GCTGTTACTGTTAGAGAATGA -3'(SEQ ID NO.122)
F2:5'-GTGTCAACCTGGTGAATAA -3'(SEQ ID NO.123)
R2:5'-GGGAAGCAGGAAGTTC -3'(SEQ ID NO.124)
P1:5'FAM-TCCGTGGACATGAGCAGCC-3'MGB (SEQ ID NO.125)
P2:5'VIC-ACCTTGCCAGCTCTGTGCC-3'MGB (SEQ ID NO.126)；
The primer and probe of 21 gene respectively has 2 groups, this two groups of primer and probe sequences are in each gene mRNA sequence The different zones design of column.
5 ' ends of the TaqMan probe are marked with fluorescent reporter group, and 3 ' ends are marked with quenching group.Same gene The reporter group of two probes must be different, quenching group can it is identical can be different.
Preferably, the fluorescent reporter group includes FAM, VIC, TET, JOE and HEX；The quenching group include BHQ1, BHQ2, TAMRA and MGB.
Preferably, corresponding two groups of primer sets for detecting same gene and probe are mixed to form primed probe premix in proportion Liquid, the kit include 21 kinds of primed probe premixed liquids for respectively corresponding 21 genes of breast cancer.
Preferably, the concentration dilution of the corresponding primer sets for detecting same gene is diluted to 50-900nM, concentration and probe concentration Primed probe premixed liquid is mixed to form after 50-500nM.
Preferably, 21 kinds of primed probe premixed liquids are attached separately in 21 QPCR reaction tubes.
It preferably, include 2 × Master Mix solution in the kit；2 × Master Mix solution includes 10-100mM Tris-Hcl、10-50 mM Kcl、 1-10 mM (NH4)2SO4、1.5-6.0mM Mgcl2、0.2-0.5mM DNTP, 0.05-0.2U HotStart Taq DNA polymerase, 10-20% glycerol and surplus ddH2O.
Second aspect, the present invention relates to a kind of detection method of 21 gene detecting kit of breast cancer, the detection methods Include the following steps:
S1, will be after 2 × Master Mix, the cDNA of sample to be tested and ddH2O mix, taking-up is added separately to 21 kinds and draws Quantitative fluorescent PCR reaction is carried out in physical prospecting needle premixed liquid；
S2, the recurrence scoring RS value that breast cancer is calculated by the CT value obtained after fluorescent quantitative PCR；RS recurs wind Danger scoring range is 0-100 points, and medium to low-risk is 0<RS<18, and medium risk is 18≤RS<31, and high risk is RS>=31.
Preferably, in step sl, the cDNA of the sample to be tested is to be prepared by a method comprising the following steps and obtain: The RNA for extracting breast cancer paraffin specimen will extract obtained RNA reverse transcription into cDNA.
Preferably, in step sl, the reaction condition of the quantitative fluorescent PCR reaction is equal are as follows: 95 DEG C of initial denaturation 10min； 95 DEG C are denaturalized 15 seconds, and 60 DEG C are annealed 30 seconds, 45-50 circulation.
Kit and detection method of the invention can be used for 21 genetic test of breast cancer, be obtained by gene expression amount calculating The recurrence of breast cancer is scored (RS), by RS value assess the risk of 10 years breast cancer relapses of therapeutic effect and prediction of individuation with And receive the benefit ratio of chemotherapy.RS risk of recurrence scoring range is 0-100 points, and medium to low-risk is 0 < RS < 18, and medium risk is 18≤RS<31, high risk are RS>=31.
Compared with prior art, the invention has the following beneficial effects:
1. kit using TaqMan probe method, contains primer and spy in the quantitative PCR system of TaqMan method Specific probes, sonde method PCR reaction compared with SYBR Green method is more special sensitiveer, and the data of acquisition are more acurrate.This reagent Box is using MGB probe, and for quenching group using non-fluorescence quenching group, itself does not generate fluorescence, can drop significantly Low background signal strength；It is connected with MGB modification group on probe simultaneously, probe Tm value is improved 10 DEG C or so, therefore than common Probe is designed shorter；It is that can inhibit the two to be combined that MGB probe is mismatched with template single base.
2. since the RNA of paraffin embedding sample is largely degradation, so 21 genes respectively devise in this kit Two pairs of primers and 2 specific probes, these two pair primer and probe are in 3 ' ends of each gene mRNA sequence and centre respectively It is designed in segment, and clip size is both less than 100bp, this provides for improved the amplification efficiencies of QPCR after degradation of rna reverse transcription；
3. two groups of primers of each gene and two probe mixed liquors are placed in a reaction tube in kit, in QPCR A kind of buffer need to be only used in amplification procedure, which simplifies experimental implementations；Each gene can be after amplification Two amplification curves are seen on QPCR, that amplification curve for then selecting amplification efficiency high obtains clear accurate CT value, makes Calculated result is more accurate credible；
4. primer and probe involved in kit, for annealing temperature all at 60 DEG C or so, this can just make 21 kinds of bases Because using same program to be expanded；TaqMan method does not need addition solubility curve, two compared with SYBR Green method simultaneously PCR can be observed directly as a result, the time of detection is greatly saved in both of which after footwork reaction；
5. kit is widely applicable, scientific research and clinical detection are all suitable for, monocyte sample or Multi-example, this can be used Kit, testing result is quick and precisely.
Detailed description of the invention
Fig. 1: the RNA electrophoretogram of test sample A, B；
Fig. 2: test sample A 21 kinds of gene QPCR amplification curve diagrams；
Two groups of CT values of Fig. 3: test sample A each gene and the RS value being calculated；
Fig. 4: test sample B 21 kinds of gene QPCR amplification curve diagrams；
Two groups of CT values of Fig. 5: test sample B each gene and the RS value being calculated；
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.
Embodiment
, paraffin sample rna extraction
After the RNA of breast cancer sample is extracted, sample to be tested A is obtained；
1) breast cancer sample is put into 1.5ml centrifuge tube；
2) 240ml PKD Buffer is added, oscillation mixes, and 11000g is centrifuged 1min；
3) 10ul Proteinase K is added, liquid-transfering gun, which is mildly inhaled, plays mixing；
4) 56 DEG C of 15min, then 80 DEG C of 15min are incubated for；
5) it is incubated for 3min on ice, 20000g is centrifuged 15min；
6) supernatant liquid is transferred to new 1.5ml centrifuge tube；
7) DNase Booster Buffer 25ul (being about centrifuged the 1/10 of liquid in pipe), and 10ul DNase is added I stock solution turns upside down mixing, and the liquid on tube wall is mixed into bottom by of short duration centrifugation；
8) it is incubated at room temperature 15min；
9) 500ul Buffer RBC is added and adjusts combining environmental, mix；
10) 1200ul dehydrated alcohol is added, mixing is played in liquid-transfering gun suction；
11) 700ul sample upper prop is drawn, >=8000g is centrifuged 15s, abandons waste liquid；
12) previous step is repeated；
13) 500ul RPE Buffer upper prop is added, >=8000g is centrifuged 15s, abandons collecting pipe waste liquid；
14) previous step is repeated, notices that waste liquid should not contact stud bottom；
15) pillar is changed in new collecting pipe, uncaps and is centrifuged 5min, abandon waste liquid；
16) pillar is changed into 1.5ml centrifuge tube, 14~30ul RNase-FNee Water, 11000g centrifugation is added 1min repeats the step to improve eluting rate, by the liquid eluted upper prop again.
17) RNA of the sample to be tested A eluted is detected into (electrophoretogram result is shown in Fig. 1 a) by electrophoresis, through detecting The RNA of sample to be tested A is complete, undegraded；
18) RNA of sample to be tested A is taken out into half, processing a period of time is carried out with RNA enzyme, after purification labeled as to be measured Sample B is detected (electrophoretogram result is shown in Fig. 1 b) by electrophoresis, and the RNA through detecting sample to be tested B is degradation.
By the RNA reverse transcription of extraction at cDNA
3. the preparation of 2 × Master Mix, primed probe premixed liquid
Its primer and probe sequence is designed according to the mRNA sequence of 21 genes, target fragment is both less than 100bp, specific sequence It arranges as follows:
ACTB:F1:5'-CCAACTTGAGATGTATGAAG -3'(SEQ ID NO.1)
R1:5'-GTCAGTGTACAGGTAAGC -3'(SEQ ID NO.2)
F2:5'-TGGAGAAGAGCTACGA -3'(SEQ ID NO.3)
R2:5'-GAAGGAAGGCTGGAAG -3'(SEQ ID NO.4)
P1:5'FAM-CTGCCTCCACCCACTCCC-3'MGB (SEQ ID NO.5)
P2:5'VIC-CGGAACCGCTCATTGCCA-3'MGB (SEQ ID NO.6)
GAPDH:F1:5'-GCCTCCAAGGAGTAAGA -3'(SEQ ID NO.7)
R1:5'-GCAGTGAGGGTCTCTC -3'(SEQ ID NO.8)
F2:5'-CCATGACAACTTTGGTATCA -3'(SEQ ID NO.9)
R2:5'-GCCATCCACAGTCTTCA -3'(SEQ ID NO.10)
P1:5'FAM-TCCTCTTGTGCTCTTGCTGG-3'MGB (SEQ ID NO.11)
P2:5'VIC-AGTCCATGCCATCACTGCCA-3'MGB (SEQ ID NO.12)
RPLPO:F1:5'--GGTTGAAGCCAAGGAAGA -3'(SEQ ID NO.13)
R1:5'-TGGTGATTAGTCAAAGAGACC -3'(SEQ ID NO.14)
F2:5'-GGGCACCATTGAAATCCA -3'(SEQ ID NO.15)
R2:5'-GGAGATGTTGAGCATGTTCA -3'(SEQ ID NO.16)
P1:5'FAM-ATCCTCGTCCGACTCCTCCGMGB-3'(SEQ ID NO.17)
P2:5'VIC-CGTGGCTTCGCTGGCTCC-3'MGB (SEQ ID NO.18)
GUS:F1:5'-CCACAGCAGCAGAACA-3'(SEQ ID NO.19)
R1:5'- CTGCTAGAATAGATGACCACAA-3'(SEQ ID NO.20)
F2:5'-GGTGTCAACAAGCATGAGA -3'(SEQ ID NO.21)
R2:5'-CAGCGAAGCAGGTTGAA -3'(SEQ ID NO.22)
P1:5'FAM-CCTCCTGGACTGTTCACGGC-3'MGB (SEQ ID NO.23)
P2:5'VIC-TCCTTCACCAGCAGCGGC-3'MGB (SEQ ID NO.24)
TFRC:F1:5'-GGACTGACATTTAGCGTAG -3'(SEQ ID NO.25)
R1:5'-GCCACATGCTTTCATTTAAG -3'(SEQ ID NO.26)
F2:5'-GAGAGGATTCCTGAGTTGAA -3'(SEQ ID NO.27)
R2:5'-TCCAGGTTCAATTCAACATCA -3'(SEQ ID NO.28)
P1:5'FAM-TGCGTAACACCCGAACCAGG-3'MGB (SEQ ID NO.29)
P2:5'VIC-CACGAACTGACCAGCGACCT-3'MGB (SEQ ID NO.30)
CD68:F1:5'-GGCAGAACTGCTTGAA -3'(SEQ ID NO.31)
R1:5'-GCGATCTTGGCTTAGTG -3'(SEQ ID NO.32)
F2:5'-ACCTGCTTCTCTCATTCCA -3'(SEQ ID NO.33)
R2:5'-CTCCACCGCCATGTAGA -3'(SEQ ID NO.34)
P1:5'FAM-CGGCTCACTGCAACCTCCA-3'MGB (SEQ ID NO.35)
P2:5'VIC-CCTTCTGCTGGAGGTCCTGC-3'MGB (SEQ ID NO.36)
BAG1:F1:5'-GCTACTACCTCTGCTTGA -3'(SEQ ID NO.37)
R1:5'-GTGAGTTGAATTCTGAAGGA -3'(SEQ ID NO.38)
F2:5'-CCACAGGCCATGAGATAA -3'(SEQ ID NO.39)
R2:5'-GGTTCTAACCTTCTGAAGACA -3'(SEQ ID NO.40)
P1:5'FAM-TCCTCACCTTCCTTCCTCCA-3'MGB (SEQ ID NO.41)
P2:5'VIC-CCGTTCCTTGGATGGAGCCT-3'MGB (SEQ ID NO.42)
GSTM1:F1:5'-CCCATCATTACCCTTCC -3'(SEQ ID NO.43)
R1:5'-GCCCTTTAAAGCAGACA -3'(SEQ ID NO.44)
F2:5'-GAGGGCTTGGAGAAGAA -3'(SEQ ID NO.45)
R2:5'-GACAGCCATCTTTGAGAAA -3'(SEQ ID NO.46)
P1:5'FAM-AGCCAGCCTGACCTTCCTTC-3'MGB (SEQ ID NO.47)
P2:5'VIC-AGTCCAGCCGCTTCCTCC-3'MGB (SEQ ID NO.48)
HER2:F1:5'-CTCCGACCACTTCCAG -3'(SEQ ID NO.49)
R1:5'-CCCTTCCAGCCATCTG -3'(SEQ ID NO.50)
F2:5'-GGTGGTTGGCATTCTGA -3'(SEQ ID NO.51)
R2:5'-CCGCATCGTGTACTTC -3'(SEQ ID NO.52)
P1:5'FAM-CTGCCATGCCAGGAACCTGT-3'MGB (SEQ ID NO.53)
P2:5'VIC-TTCTGCTGCCGTCGCTTGA-3'MGB (SEQ ID NO.54)
GRB7:F1:5'-CCTCCCTCAGATAGAA -3'(SEQ ID NO.55)
R1:5'-GCTAGGAGAAGGAGAG -3'(SEQ ID NO.56)
F2:5'-GGCTTCGGATCTTCTGA -3'(SEQ ID NO.57)
R2:5'-CCGTACTTGAAGAGGC -3'(SEQ ID NO.58)
P1:5'FAM-CCACTCCAGTCCACTCCTGA-3'MGB (SEQ ID NO.59)
P2:5'VIC-AGATGAGCAGAGCCGCACC-3'MGB (SEQ ID NO.60)
MMP11:F1:5'-AGGTCAGGTCTTGGTA -3'(SEQ ID NO.61)
R1:5'-CTGGATTCTGGAGAACA -3'(SEQ ID NO.62)
F2:5'--CCAGATGCCTGTGAGGA -3'(SEQ ID NO.63)
R2:5'-AGCCCGCTTTGAAGAA -3'(SEQ ID NO.64)
P1:5'FAM TTCTGGCTGACAATCCTGGA-3'MGB (SEQ ID NO.65)
P2:5'VIC-TCCACCATCCGAGGCGAGC-3'MGB (SEQ ID NO.66)
CTSL2:F1:5'-CCTAGATGGTATAGCCTATTAC -3'(SEQ ID NO.67)
R1:5'-AGCACAGTGATGTGTTG -3'(SEQ ID NO.68)
F2:5'-GGCTGGTCTTGAACTCA -3'(SEQ ID NO.69)
R2:5'-TGGCTCACATCTGTAATC -3'(SEQ ID NO.70)
P1:5'FAM-TGGTACAGCCTGTTGCTCCT-3'MGB (SEQ ID NO.71)
P2:5'VIC-CCACCTGCCTCAGCCTCC-3'MGB (SEQ ID NO.72)
KI67:F1:5'-CTCTGTACCTACCATCTCC -3'(SEQ ID NO.73)
R1:5'-CAGGAGGGAAAGTTCCA -3'(SEQ ID NO.74)
F2:5'-GGGAAAGTAGGTGTGAAAGA -3'(SEQ ID NO.75)
R2:5'-CCATCTCCTGCTGTCTC -3'(SEQ ID NO.76)
P1:5'FAM-AGGCACCTCCAACCACCACA-3'MGB (SEQ ID NO.77)
P2:5'VIC-ACCAGTCGGCAAGCTCACAC-3'MGB (SEQ ID NO.78)
STK15:F1:5'-ACAGGAGGCAAATCC -3'(SEQ ID NO.79)
R1:5'-CAGGTACTAGGAAGGTTATTG -3'(SEQ ID NO.80)
F2:5'-GGGTGGTCAGTACATGA -3'(SEQ ID NO.81)
R2:5'-CATCCGACCTTCAATCA -3'(SEQ ID NO.82)
P1:5'FAM-TGACCACTCTGCCCTGACCC-3'MGB (SEQ ID NO.83)
P2:5'VIC-TCCATCTTCCAGGAGGACCA-3'MGB (SEQ ID NO.84)
CCNB1:F1:5'-CTGTCAAGAACAAGTATGC -3'(SEQ ID NO.85)
R1:5'-CACAGCCTTGGCTAAA -3'(SEQ ID NO.86)
F2:5'-ATCGGTTCATGCAGAATAATTGA -3'(SEQ ID NO.87)
R2:5'-GGAGGGTACATTTCTTCATATTTG -3'(SEQ ID NO.88)
P1:5'FAM-AAGATCAGCACTCTACCACAGC-3'MGB (SEQ ID NO.89)
P2:5'VIC-TGGCAGTGACACCAACCAGC-3'MGB (SEQ ID NO.90)
MYBL2:F1:5'-CAGACTCTCAGGTGGA -3'(SEQ ID NO.91)
R1:5'-GACCTGGAAGTGGAAC -3'(SEQ ID NO.92)
F2:5'-GCCCTCTGTGCTCAAGA -3'(SEQ ID NO.93)
R2:5'-CAGACTGGTGCTATTCTCA -3'(SEQ ID NO.94)
P1:5'FAM-AGCACCAGGAGCCGCC-3'MGB (SEQ ID NO.95)
P2:5'VIC-CACACGCCTCTTCCTCTGCC-3'MGB (SEQ ID NO.96)
Survivin:F1:5'-GCCATCCTTAAAACCAGA -3'(SEQ ID NO.97)
R1:5'-CGGTTGCACTTAGACAA -3'(SEQ ID NO.98)
F2:5'-CCTTCACATCTGTCACGA -3'(SEQ ID NO.99)
R2:5'-GCTGCCTCCAAAGAAAG -3'(SEQ ID NO.100)
P1:5'FAM-CCTCATGGCTACCAGCACCT-3'MGB (SEQ ID NO.101)
P2:5'VIC-ACGCAGTCCGCCCAGGT-3'MGB (SEQ ID NO.102)
ESR1:F1:5'-GCACCCTAGAAACAACATA -3'(SEQ ID NO.103)
R1:5'-GTCTCTATAACCAATGACCTC -3'(SEQ ID NO.104)
F2:5'-CTGGTGATTGTCAATTCATTCA -3'(SEQ ID NO.105)
R2:5'-GCAGTGTATCAAGTTAAAATAGTC -3'(SEQ ID NO.106)
P1:5'FAM-AGGTGCCTGAGACACAGACC-3'MGB (SEQ ID NO.107)
P2:5'VIC-TCTGCCTTCCCTGTGTGCCC-3'MGB (SEQ ID NO.108)
PGR:F1:5'-TGCCAAGAAGGTGAAAC -3'(SEQ ID NO.109)
R1:5'-ACAGCTTTGCATTGTCA -3'(SEQ ID NO.110)
F2:5'-CATGGAATGTCAACCTGTAA -3'(SEQ ID NO.111)
R2:5'-GCACAGTCCTATGTCAG -3'(SEQ ID NO.112)
P1:5'FAM-CCATCACTGCCAGGGACCCA-3'MGB (SEQ ID NO.113)
P2:5'VIC-CTCCACAGTGCCCATCATCA-3'MGB (SEQ ID NO.114)
BCL2:F1:5'-GGGTATGAAGGACCTGTA -3'(SEQ ID NO.115)
R1:5'- GTCGTACCACAAACTTCAAA-3'(SEQ ID NO.116)
F2:5'-GTGAGGAGAGGCAATGA -3'(SEQ ID NO.117)
R2:5'-CCTTGCTTTAAACTCACAG -3'(SEQ ID NO.118)
P1:5'FAM-TGCCTCTGCGAAGAACCTTG-3'MGB (SEQ ID NO.119)
P2:5'VIC-CAGCCAACGTGCCATGTGC-3'MGB (SEQ ID NO.120)
SCUBE2:F1:5'-CCCAGTCTCAAATGTGA -3'(SEQ ID NO.121)
R1:5'-GCTGTTACTGTTAGAGAATGA -3'(SEQ ID NO.122)
F2:5'-GTGTCAACCTGGTGAATAA -3'(SEQ ID NO.123)
R2:5'-GGGAAGCAGGAAGTTC -3'(SEQ ID NO.124)
P1:5'FAM-TCCGTGGACATGAGCAGCC-3'MGB (SEQ ID NO.125)
P2:5'VIC-ACCTTGCCAGCTCTGTGCC-3'MGB (SEQ ID NO.126)
2) preparation of reagent
A. the preparation of 2 × Master Mix
2 × Master Mix component Concentration range
Tris-Hcl(pH8.0-pH9.0) 10-100mM
Kcl 10-50 mM
(NH4)2SO4 1-10 mM
Mgcl2 1.5-6.0mM
dNTP 0.2-0.5mM
HotStart Taq DNA polymerase 0.05-0.2U
Glycerol 10-20%
ddH2O In right amount
B. by the concentration dilution of 42 pairs of primers to 50-900nM；
C. 42 concentration and probe concentrations are diluted to 50-500nM；
D. primed probe premixed liquid is prepared；
Respectively according to a certain percentage (F:R:P=3:3:2) by two pairs of primers of each gene in 21 genes and two probes It is mixed, the bottom that 2 μ l are added to 21 QPCR reaction tubes is taken out after mixing, closes the lid, aluminium foil plastic packaging is put into after centrifugation Bag, takes out to the used time, and others are placed on -20 DEG C of preservations.
The preparation of reaction solution
Reaction system (10 μ L)
4.1 directly take out 2 × Master Mix and primed probe premixed liquid from kit, and are centrifuged；
4.2 take out the sample to be tested cDNA after reverse transcription；
4.3 calculate and need the stoichiometric number that detects, by reaction system take out corresponding amount 2 × Master Mix, ddH2O and Sample to be tested cDNA, is then mixed well, and centrifugation, each reaction tube dispenses 8 μ l；
4.4 cover the lid of reaction tube, have to cover tightly lid and (prevent from evaporating during PCR), are then centrifuged for counting Second；
4.5 open fluorescence quantitative PCR instrument, and the reaction tube added is placed on fluorescence quantitative PCR instrument.
Amplification program
95℃ 10min
6.QPCR testing result
RS value is calculated by the amplification curve that obtains after fluorescent quantitative PCR and resulting CT value.
1. the detection (such as Fig. 2, Fig. 3) of sample to be tested A:
As shown, two groups of primer and probes of 21 genes of sample to be tested A have amplification curve and CT value, and 2 times It is close to repeat CT value.Each gene has 2 groups of data, what wherein CT value was small be classified as TEST1, CT value it is larger be classified as TEST2. The RS value for being computed TEST1 is 24.431,18≤RS < 31, belongs to moderate risk, and the RS value of TEST2 is 24.436,18≤RS < 31, belong to moderate risk.It is undegraded since the RNA of the sample is complete, so the RS value difference of two groups of data is not little, basic one It causes.
2. the detection (such as Fig. 4, Fig. 5) of sample to be tested B:
As shown, two groups of primer and probes of 21 genes of sample to be tested B have amplification curve and CT value, and 2 times It is close to repeat CT value.Each gene has 2 groups of data, what wherein CT value was small be classified as TEST1, CT value it is larger be classified as TEST2. The RS value for being computed TEST1 is 24.431, and 18≤RS < 31 belong to moderate risk, consistent with the testing result of sample to be tested A； The RS value of TEST2 is 33.45, RS > 31, belongs to high risk, inconsistent with the testing result of sample to be tested A, this is because the sample This RNA has degraded, so the RS value difference that two groups of data calculate is larger.It follows that this kit is highly suitable for dropping Solve RNA.
The sequencing of above embodiments is not only for ease of description, represent the advantages or disadvantages of the embodiments.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features； And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and Range.
Sequence table
<110>Mo He medical technology (Shanghai) Co., Ltd.
<120>a kind of breast cancer 21 is based on detection kit and its detection method
<160> 126
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ccaacttgag atgtatgaag 20
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gtcagtgtac aggtaagc 18
<210> 3
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tggagaagag ctacga 16
<210> 4
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gaaggaaggc tggaag 16
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ctgcctccac ccactccc 18
<210> 6
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cggaaccgct cattgcca 18
<210> 7
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gcctccaagg agtaaga 17
<210> 8
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gcagtgaggg tctctc 16
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ccatgacaac tttggtatca 20
<210> 10
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gccatccaca gtcttca 17
<210> 11
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tcctcttgtg ctcttgctgg 20
<210> 12
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
agtccatgcc atcactgcca 20
<210> 13
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
ggttgaagcc aaggaaga 18
<210> 14
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
tggtgattag tcaaagagac c 21
<210> 15
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
gggcaccatt gaaatcca 18
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
ggagatgttg agcatgttca 20
<210> 17
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
atcctcgtcc gactcctccg 20
<210> 18
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
cgtggcttcg ctggctcc 18
<210> 19
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
ccacagcagc agaaca 16
<210> 20
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ctgctagaat agatgaccac aa 22
<210> 21
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ggtgtcaaca agcatgaga 19
<210> 22
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
cagcgaagca ggttgaa 17
<210> 23
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
cctcctggac tgttcacggc 20
<210> 24
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
tccttcacca gcagcggc 18
<210> 25
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
ggactgacat ttagcgtag 19
<210> 26
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
gccacatgct ttcatttaag 20
<210> 27
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
gagaggattc ctgagttgaa 20
<210> 28
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
tccaggttca attcaacatc a 21
<210> 29
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
tgcgtaacac ccgaaccagg 20
<210> 30
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
cacgaactga ccagcgacct 20
<210> 31
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
ggcagaactg cttgaa 16
<210> 32
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
gcgatcttgg cttagtg 17
<210> 33
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
acctgcttct ctcattcca 19
<210> 34
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
ctccaccgcc atgtaga 17
<210> 36
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
cggctcactg caacctcca 19
<210> 37
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
ccttctgctg gaggtcctgc 20
<210> 38
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
gctactacct ctgcttga 18
<210> 39
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
gtgagttgaa ttctgaagga 20
<210> 40
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
ccacaggcca tgagataa 18
<210> 41
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
ggttctaacc ttctgaagac a 21
<210> 42
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
tcctcacctt ccttcctcca 20
<210> 42
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
ccgttccttg gatggagcct 20
<210> 43
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
cccatcatta cccttcc 17
<210> 45
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
gccctttaaa gcagaca 17
<210> 46
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
gagggcttgg agaagaa 17
<210> 47
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
gacagccatc tttgagaaa 19
<210> 47
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
agccagcctg accttccttc 20
<210> 48
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
agtccagccg cttcctcc 18
<210> 49
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
ctccgaccac ttccag 16
<210> 50
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
cccttccagc catctg 16
<210> 51
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
ggtggttggc attctga 17
<210> 52
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
ccgcatcgtg tacttc 16
<210> 53
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
ctgccatgcc aggaacctgt 20
<210> 54
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
ttctgctgcc gtcgcttga 19
<210> 55
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
cctccctcag atagaa 16
<210> 56
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
gctaggagaa ggagag 16
<210> 57
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
ggcttcggat cttctga 17
<210> 58
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
ccgtacttga agaggc 16
<210> 59
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
ccactccagt ccactcctga 20
<210> 60
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
agatgagcag agccgcacc 19
<210> 61
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
aggtcaggtc ttggta 16
<210> 62
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
ctggattctg gagaaca 17
<210> 63
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
ccagatgcct gtgagga 17
<210> 64
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
agcccgcttt gaagaa 16
<210> 65
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
ttctggctga caatcctgga 20
<210> 66
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
tccaccatcc gaggcgagc 19
<210> 67
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
cctagatggt atagcctatt ac 22
<210> 68
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
agcacagtga tgtgttg 17
<210> 69
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
ggctggtctt gaactca 17
<210> 70
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
tggctcacat ctgtaatc 18
<210> 71
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
tggtacagcc tgttgctcct 20
<210> 72
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
ccacctgcct cagcctcc 18
<210> 73
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
ctctgtacct accatctcc 19
<210> 74
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
caggagggaa agttcca 17
<210> 75
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
gggaaagtag gtgtgaaaga 20
<210> 76
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
ccatctcctg ctgtctc 17
<210> 77
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
aggcacctcc aaccaccaca 20
<210> 78
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
accagtcggc aagctcacac 20
<210> 79
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 79
acaggaggca aatcc 15
<210> 80
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 80
caggtactag gaaggttatt g 21
<210> 81
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 81
gggtggtcag tacatga 17
<210> 82
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 82
catccgacct tcaatca 17
<210> 83
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 83
tgaccactct gccctgaccc 20
<210> 84
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 84
tccatcttcc aggaggacca 20
<210> 85
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 85
ctgtcaagaa caagtatgc 19
<210> 86
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 86
cacagccttg gctaaa 16
<210> 87
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 87
atcggttcat gcagaataat tga 23
<210> 88
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 88
ggagggtaca tttcttcata tttg 24
<210> 89
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 89
aagatcagca ctctaccaca gc 22
<210> 90
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 90
tggcagtgac accaaccagc 20
<210> 91
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 91
cagactctca ggtgga 16
<210> 92
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 92
gacctggaag tggaac 16
<210> 93
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 93
gccctctgtg ctcaaga 17
<210> 94
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 94
cagactggtg ctattctca 19
<210> 95
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 95
agcaccagga gccgcc 16
<210> 96
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 96
cacacgcctc ttcctctgcc 20
<210> 97
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 97
gccatcctta aaaccaga 18
<210> 98
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 98
cggttgcact tagacaa 17
<210> 99
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 99
ccttcacatc tgtcacga 18
<210> 100
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 100
gctgcctcca aagaaag 17
<210> 101
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 101
cctcatggct accagcacct 20
<210> 102
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 102
acgcagtccg cccaggt 17
<210> 103
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 103
gcaccctaga aacaacata 19
<210> 104
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 104
gtctctataa ccaatgacct c 21
<210> 105
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 105
ctggtgattg tcaattcatt ca 22
<210> 106
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 106
gcagtgtatc aagttaaaat agtc 24
<210> 107
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 107
aggtgcctga gacacagacc 20
<210> 108
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 108
tctgccttcc ctgtgtgccc 20
<210> 109
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 109
tgccaagaag gtgaaac 17
<210> 110
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 110
acagctttgc attgtca 17
<210> 111
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 111
catggaatgt caacctgtaa 20
<210> 112
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 112
gcacagtcct atgtcag 17
<210> 113
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 113
ccatcactgc cagggaccca 20
<210> 114
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 114
ctccacagtg cccatcatca 20
<210> 115
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 115
gggtatgaag gacctgta 18
<210> 116
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 116
gtcgtaccac aaacttcaaa 20
<210> 117
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 117
gtgaggagag gcaatga 17
<210> 118
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 118
ccttgcttta aactcacag 19
<210> 119
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 119
tgcctctgcg aagaaccttg 20
<210> 120
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 120
cagccaacgt gccatgtgc 19
<210> 121
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 121
cccagtctca aatgtga 17
<210> 122
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 122
gctgttactg ttagagaatg a 21
<210> 123
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 123
gtgtcaacct ggtgaataa 19
<210> 124
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 124
gggaagcagg aagttc 16
<210> 125
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 125
tccgtggaca tgagcagcc 19
<210> 126
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 126
accttgccag ctctgtgcc 19

Claims (6)

1. a kind of 21 gene detecting kit of breast cancer, it is characterized in that 21 genes are as follows: ACTB, GAPDH, RPLPO, GUS, TFRC、CD68、BAG1、GSTM1、HER2、GRB7、MMP11、CTSL2、KI67、STK15、CCNB1、MYBL2、Survivin、 ESR1, PGR, BCL2 and SCUBE2；
Two pairs of primers and 2 specific probes, primer corresponding to 21 genes are respectively devised for 21 genes in kit Sequence and TaqMan probe sequence difference are as follows:
(1) primer sequence of ACTB gene is detected are as follows: SEQ ID NO.1 and SEQ ID NO.2 and SEQ ID NO.3 and SEQ ID NO.4 detects the TaqMan probe sequence of ACTB gene are as follows: SEQ ID NO.5 and SEQ ID NO.6；Particular sequence is as follows:
ACTB:
F1:5'-CCAACTTGAGATGTATGAAG -3'(SEQ ID NO.1)
R1:5'-GTCAGTGTACAGGTAAGC -3'(SEQ ID NO.2)
F2:5'-TGGAGAAGAGCTACGA -3'(SEQ ID NO.3)
R2:5'-GAAGGAAGGCTGGAAG -3'(SEQ ID NO.4)
P1:5'FAM-CTGCCTCCACCCACTCCC-3'MGB (SEQ ID NO.5)
P2:5'VIC-CGGAACCGCTCATTGCCA-3'MGB (SEQ ID NO.6)；
(2) primer sequence of GAPDH gene is detected are as follows: SEQ ID NO.7 and SEQ ID NO.8 and SEQ ID NO.9 and SEQ ID NO.10 detects the TaqMan probe sequence of GAPDH gene are as follows: SEQ ID NO.11 and SEQ ID NO.12；Particular sequence It is as follows:
GAPDH:
F1:5'-GCCTCCAAGGAGTAAGA -3'(SEQ ID NO.7)
R1:5'-GCAGTGAGGGTCTCTC -3'(SEQ ID NO.8)
F2:5'-CCATGACAACTTTGGTATCA -3'(SEQ ID NO.9)
R2:5'-GCCATCCACAGTCTTCA -3'(SEQ ID NO.10)
P1:5'FAM-TCCTCTTGTGCTCTTGCTGG-3'MGB (SEQ ID NO.11)
P2:5'VIC-AGTCCATGCCATCACTGCCA-3'MGB (SEQ ID NO.12)；
(3) detect RPLPO gene primer sequence are as follows: SEQ ID NO.13 and SEQ ID NO.14 and SEQ ID NO.15 and SEQ ID NO.16 detects the TaqMan probe sequence of RPLPO gene are as follows: SEQ ID NO.17 and SEQ ID NO.18；Specifically Sequence is as follows:
RPLPO:
F1:5'--GGTTGAAGCCAAGGAAGA -3'(SEQ ID NO.13)
R1:5'-TGGTGATTAGTCAAAGAGACC -3'(SEQ ID NO.14)
F2:5'-GGGCACCATTGAAATCCA -3'(SEQ ID NO.15)
R2:5'-GGAGATGTTGAGCATGTTCA -3'(SEQ ID NO.16)
P1:5'FAM-ATCCTCGTCCGACTCCTCCG-3'MGB (SEQ ID NO.17)
P2:5'VIC-CGTGGCTTCGCTGGCTCC-3'MGB (SEQ ID NO.18)；
(4) primer sequence of gus gene is detected are as follows: SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22 detects the TaqMan probe sequence of gus gene are as follows: SEQ ID NO.23 and SEQ ID NO.24；Particular sequence is such as Under:
GUS:
F1:5'-CCACAGCAGCAGAACA-3'(SEQ ID NO.19)
R1:5'- CTGCTAGAATAGATGACCACAA-3'(SEQ ID NO.20)
F2:5'-GGTGTCAACAAGCATGAGA -3'(SEQ ID NO.21)
R2:5'-CAGCGAAGCAGGTTGAA -3'(SEQ ID NO.22)
P1:5'FAM-CCTCCTGGACTGTTCACGGC-3'MGB (SEQ ID NO.23)
P2:5'VIC-TCCTTCACCAGCAGCGGC-3'MGB (SEQ ID NO.24)；
(5) primer sequence of TFRC gene is detected are as follows: SEQ ID NO.25 and SEQ ID NO.26 and SEQ ID NO.27 and SEQ ID NO.28 detects the TaqMan probe sequence of TFRC gene are as follows: SEQ ID NO.29 and SEQ ID NO.30；Particular sequence is such as Under:
TFRC:
F1:5'-GGACTGACATTTAGCGTAG -3'(SEQ ID NO.25)
R1:5'-GCCACATGCTTTCATTTAAG -3'(SEQ ID NO.26)
F2:5'-GAGAGGATTCCTGAGTTGAA -3'(SEQ ID NO.27)
R2:5'-TCCAGGTTCAATTCAACATCA -3'(SEQ ID NO.28)
P1:5'FAM-TGCGTAACACCCGAACCAGG-3'MGB (SEQ ID NO.29)
P2:5'VIC-CACGAACTGACCAGCGACCT-3'MGB (SEQ ID NO.30)；
(6) primer sequence for detecting CD68 gene is SEQ ID NO.31 and SEQ ID NO.32 and SEQ ID NO.33 and SEQ ID NO.34 detects the TaqMan probe sequence of CD68 gene are as follows: SEQ ID NO.35 and SEQ ID NO.36；Particular sequence is such as Under:
CD68:
F1:5'-GGCAGAACTGCTTGAA -3'(SEQ ID NO.31)
R1:5'-GCGATCTTGGCTTAGTG -3'(SEQ ID NO.32)
F2:5'-ACCTGCTTCTCTCATTCCA -3'(SEQ ID NO.33)
R2:5'-CTCCACCGCCATGTAGA -3'(SEQ ID NO.34)
P1:5'FAM-CGGCTCACTGCAACCTCCA-3'MGB (SEQ ID NO.35)
P2:5'VIC-CCTTCTGCTGGAGGTCCTGC-3'MGB (SEQ ID NO.36)；
(7) primer sequence of BAG1 gene is detected are as follows: SEQ ID NO.37 and SEQ ID NO.38 and SEQ ID NO.39 and SEQ ID NO.40 detects the TaqMan probe sequence of BAG1 gene are as follows: SEQ ID NO.41 and SEQ ID NO.42；Particular sequence is such as Under:
BAG1:
F1:5'-GCTACTACCTCTGCTTGA -3'(SEQ ID NO.37)
R1:5'-GTGAGTTGAATTCTGAAGGA -3'(SEQ ID NO.38)
F2:5'-CCACAGGCCATGAGATAA -3'(SEQ ID NO.39)
R2:5'-GGTTCTAACCTTCTGAAGACA -3'(SEQ ID NO.40)
P1:5'FAM-TCCTCACCTTCCTTCCTCCA-3'MGB (SEQ ID NO.41)
P2:5'VIC-CCGTTCCTTGGATGGAGCCT-3'MGB (SEQ ID NO.42)；
(8) detect GSTM1 gene primer sequence are as follows: SEQ ID NO.43 and SEQ ID NO.44 and SEQ ID NO.45 and SEQ ID NO.46 detects the TaqMan probe sequence of GSTM1 gene are as follows: SEQ ID NO.47 and SEQ ID NO.48；Specifically Sequence is as follows:
GSTM1:
F1:5'-CCCATCATTACCCTTCC -3'(SEQ ID NO.43)
R1:5'-GCCCTTTAAAGCAGACA -3'(SEQ ID NO.44)
F2:5'-GAGGGCTTGGAGAAGAA -3'(SEQ ID NO.45)
R2:5'-GACAGCCATCTTTGAGAAA -3'(SEQ ID NO.46)
P1:5'FAM-AGCCAGCCTGACCTTCCTTC-3'MGB (SEQ ID NO.47)
P2:5'VIC-AGTCCAGCCGCTTCCTCC-3'MGB (SEQ ID NO.48)；
(9) primer sequence of HER2 gene is detected are as follows: SEQ ID NO.49 and SEQ ID NO.50 and SEQ ID NO.51 and SEQ ID NO.52 detects the TaqMan probe sequence of HER2 gene are as follows: SEQ ID NO.53 and SEQ ID NO.54；Particular sequence is such as Under:
HER2:
F1:5'-CTCCGACCACTTCCAG -3'(SEQ ID NO.49)
R1:5'-CCCTTCCAGCCATCTG -3'(SEQ ID NO.50)
F2:5'-GGTGGTTGGCATTCTGA -3'(SEQ ID NO.51)
R2:5'-CCGCATCGTGTACTTC -3'(SEQ ID NO.52)
P1:5'FAM-CTGCCATGCCAGGAACCTGT-3'MGB (SEQ ID NO.53)
P2:5'VIC-TTCTGCTGCCGTCGCTTGA-3'MGB (SEQ ID NO.54)；
(10) detect GRB7 gene primer sequence are as follows: SEQ ID NO.55 and SEQ ID NO.56 and SEQ ID NO.57 and SEQ ID NO.58 detects the TaqMan probe sequence of GRB7 gene are as follows: SEQ ID NO.59 and SEQ ID NO.60；Specific sequence It arranges as follows:
GRB7:
F1:5'-CCTCCCTCAGATAGAA -3'(SEQ ID NO.55)
R1:5'-GCTAGGAGAAGGAGAG -3'(SEQ ID NO.56)
F2:5'-GGCTTCGGATCTTCTGA -3'(SEQ ID NO.57)
R2:5'-CCGTACTTGAAGAGGC -3'(SEQ ID NO.58)
P1:5'FAM-CCACTCCAGTCCACTCCTGA-3'MGB (SEQ ID NO.59)
P2:5'VIC-AGATGAGCAGAGCCGCACC-3'MGB (SEQ ID NO.60)；
(11) detect MMP11 gene primer sequence are as follows: SEQ ID NO.61 and SEQ ID NO.62 and SEQ ID NO.63 and SEQ ID NO.64 detects the TaqMan probe sequence of MMP11 gene are as follows: SEQ ID NO.65 and SEQ ID NO.66；Specifically Sequence is as follows:
MMP11:
F1:5'-AGGTCAGGTCTTGGTA -3'(SEQ ID NO.61)
R1:5'-CTGGATTCTGGAGAACA -3'(SEQ ID NO.62)
F2:5'--CCAGATGCCTGTGAGGA -3'(SEQ ID NO.63)
R2:5'-AGCCCGCTTTGAAGAA -3'(SEQ ID NO.64)
P1:5'FAM-TTCTGGCTGACAATCCTGGA-3'MGB (SEQ ID NO.65)
P2:5'VIC-TCCACCATCCGAGGCGAGC-3'MGB (SEQ ID NO.66)；
(12) detect CTSL2 gene primer sequence are as follows: SEQ ID NO.67 and SEQ ID NO.68 and SEQ ID NO.69 and SEQ ID NO.70；Detect the TaqMan probe sequence of CTSL2 gene are as follows: SEQ ID NO.71 and SEQ ID NO.72；Specifically Sequence is as follows:
CTSL2:
F1:5'-CCTAGATGGTATAGCCTATTAC -3'(SEQ ID NO.67)
R1:5'-AGCACAGTGATGTGTTG -3'(SEQ ID NO.68)
F2:5'-GGCTGGTCTTGAACTCA -3'(SEQ ID NO.69)
R2:5'-TGGCTCACATCTGTAATC -3'(SEQ ID NO.70)
P1:5'FAM-TGGTACAGCCTGTTGCTCCT-3'MGB (SEQ ID NO.71)
P2:5'VIC-CCACCTGCCTCAGCCTCC-3'MGB (SEQ ID NO.72)；
(13) detect KI67 gene primer sequence are as follows: SEQ ID NO.73 and SEQ ID NO.74 and SEQ ID NO.75 and SEQ ID NO.76 detects the TaqMan probe sequence of KI67 gene are as follows: SEQ ID NO.77 and SEQ ID NO.78；Specific sequence It arranges as follows:
KI67:
F1:5'-CTCTGTACCTACCATCTCC -3'(SEQ ID NO.73)
R1:5'-CAGGAGGGAAAGTTCCA -3'(SEQ ID NO.74)
F2:5'-GGGAAAGTAGGTGTGAAAGA -3'(SEQ ID NO.75)
R2:5'-CCATCTCCTGCTGTCTC -3'(SEQ ID NO.76)
P1:5'FAM-AGGCACCTCCAACCACCACA-3'MGB (SEQ ID NO.77)
P2:5'VIC-ACCAGTCGGCAAGCTCACAC-3'MGB (SEQ ID NO.78)；
(14) detect STK15 gene primer sequence are as follows: SEQ ID NO.79 and SEQ ID NO.80 and SEQ ID NO.81 and SEQ ID NO.82 detects the TaqMan probe sequence of STK15 gene are as follows: SEQ ID NO.83 and SEQ ID NO.84；Specifically Sequence is as follows:
STK15:
F1:5'-ACAGGAGGCAAATCC -3'(SEQ ID NO.79)
R1:5'-CAGGTACTAGGAAGGTTATTG -3'(SEQ ID NO.80)
F2:5'-GGGTGGTCAGTACATGA -3'(SEQ ID NO.81)
R2:5'-CATCCGACCTTCAATCA -3'(SEQ ID NO.82)
P1:5'FAM-TGACCACTCTGCCCTGACCC-3'MGB (SEQ ID NO.83)
P2:5'VIC-TCCATCTTCCAGGAGGACCA-3'MGB (SEQ ID NO.84)；
(15) detect CCNB1 gene primer sequence are as follows: SEQ ID NO.85 and SEQ ID NO.86 and SEQ ID NO.87 and SEQ ID NO.88 detects the TaqMan probe sequence of CCNB1 gene are as follows: SEQ ID NO.89 and SEQ ID NO.90；Specifically Sequence is as follows:
CCNB1:
F1:5'-CTGTCAAGAACAAGTATGC -3'(SEQ ID NO.85)
R1:5'-CACAGCCTTGGCTAAA -3'(SEQ ID NO.86)
F2:5'-ATCGGTTCATGCAGAATAATTGA -3'(SEQ ID NO.87)
R2:5'-GGAGGGTACATTTCTTCATATTTG -3'(SEQ ID NO.88)
P1:5'FAM-AAGATCAGCACTCTACCACAGC-3'MGB (SEQ ID NO.89)
P2:5'VIC-TGGCAGTGACACCAACCAGC-3'MGB (SEQ ID NO.90)；
(16) detect MYBL2 gene primer sequence are as follows: SEQ ID NO.91 and SEQ ID NO.92 and SEQ ID NO.93 and SEQ ID NO.94 detects the TaqMan probe sequence of MYBL2 gene are as follows: SEQ ID NO.95 and SEQ ID NO.96；Specifically Sequence is as follows:
MYBL2:
F1:5'-CAGACTCTCAGGTGGA -3'(SEQ ID NO.91)
R1:5'-GACCTGGAAGTGGAAC -3'(SEQ ID NO.92)
F2:5'-GCCCTCTGTGCTCAAGA -3'(SEQ ID NO.93)
R2:5'-CAGACTGGTGCTATTCTCA -3'(SEQ ID NO.94)
P1:5'FAM-AGCACCAGGAGCCGCC-3'MGB (SEQ ID NO.95)
P2:5'VIC-CACACGCCTCTTCCTCTGCC-3'MGB (SEQ ID NO.96)；
(17) primer sequence of Survivin gene is detected are as follows: SEQ ID NO.97 and SEQ ID NO.98 and SEQ ID NO.99 With SEQ ID NO.100, the TaqMan probe sequence for detecting Survivin gene is SEQ ID NO.101 and SEQ ID NO.102；Particular sequence is as follows:
Survivin:
F1:5'-GCCATCCTTAAAACCAGA -3'(SEQ ID NO.97)
R1:5'-CGGTTGCACTTAGACAA -3'(SEQ ID NO.98)
F2:5'-CCTTCACATCTGTCACGA -3'(SEQ ID NO.99)
R2:5'-GCTGCCTCCAAAGAAAG -3'(SEQ ID NO.100)
P1:5'FAM-CCTCATGGCTACCAGCACCT-3'MGB (SEQ ID NO.101)
P2:5'VIC-ACGCAGTCCGCCCAGGT-3'MGB (SEQ ID NO.102)；
(18) primer sequence of ESR1 gene is detected are as follows: SEQ ID NO.103 and SEQ ID NO.104 and SEQ ID NO.105 With SEQ ID NO.106, the TaqMan probe sequence of ESR1 gene is detected are as follows: SEQ ID NO.107 and SEQ ID NO.108； Particular sequence is as follows:
ESR1:
F1:5'-GCACCCTAGAAACAACATA -3'(SEQ ID NO.103)
R1:5'-GTCTCTATAACCAATGACCTC -3'(SEQ ID NO.104)
F2:5'-CTGGTGATTGTCAATTCATTCA -3'(SEQ ID NO.105)
R2:5'-GCAGTGTATCAAGTTAAAATAGTC -3'(SEQ ID NO.106)
P1:5'FAM-AGGTGCCTGAGACACAGACC-3'MGB (SEQ ID NO.107)
P2:5'VIC-TCTGCCTTCCCTGTGTGCCC-3'MGB (SEQ ID NO.108)；
(19) detect PGR gene primer sequence are as follows: SEQ ID NO.109 and SEQ ID NO.110 and SEQ ID NO.111 and SEQ ID NO.112 detects the TaqMan probe sequence of PGR gene are as follows: SEQ ID NO.113 and SEQ ID NO.114；Specifically Sequence is as follows:
PGR:
F1:5'-TGCCAAGAAGGTGAAAC -3'(SEQ ID NO.109)
R1:5'-ACAGCTTTGCATTGTCA -3'(SEQ ID NO.110)
F2:5'-CATGGAATGTCAACCTGTAA -3'(SEQ ID NO.111)
R2:5'-GCACAGTCCTATGTCAG -3'(SEQ ID NO.112)
P1:5'FAM-CCATCACTGCCAGGGACCCA-3'MGB (SEQ ID NO.113)
P2:5'VIC-CTCCACAGTGCCCATCATCA-3'MGB (SEQ ID NO.114)；
(20) primer sequence of BCL2 gene is detected are as follows: SEQ ID NO.115 and SEQ ID NO.116 and SEQ ID NO.117 With SEQ ID NO.118, the TaqMan probe sequence of BCL2 gene is detected are as follows: SEQ ID NO.119 and SEQ ID NO.120； Particular sequence is as follows:
BCL2:
F1:5'-GGGTATGAAGGACCTGTA -3'(SEQ ID NO.115)
R1:5'- GTCGTACCACAAACTTCAAA-3'(SEQ ID NO.116)
F2:5'-GTGAGGAGAGGCAATGA -3'(SEQ ID NO.117)
R2:5'-CCTTGCTTTAAACTCACAG -3'(SEQ ID NO.118)
P1:5'FAM-TGCCTCTGCGAAGAACCTTG-3'MGB (SEQ ID NO.119)
P2:5'VIC-CAGCCAACGTGCCATGTGC-3'MGB (SEQ ID NO.120)；
(21) primer sequence of SCUBE2 gene is detected are as follows: SEQ ID NO.121 and SEQ ID NO.122 and SEQ ID NO.123 and SEQ ID NO.124 detects the TaqMan probe sequence of SCUBE2 gene are as follows: SEQ ID NO.125 and SEQ ID NO.126；Particular sequence is as follows:
SCUBE2:
F1:5'-CCCAGTCTCAAATGTGA -3'(SEQ ID NO.121)
R1:5'-GCTGTTACTGTTAGAGAATGA -3'(SEQ ID NO.122)
F2:5'-GTGTCAACCTGGTGAATAA -3'(SEQ ID NO.123)
R2:5'-GGGAAGCAGGAAGTTC -3'(SEQ ID NO.124)
P1:5'FAM-TCCGTGGACATGAGCAGCC-3'MGB (SEQ ID NO.125)
P2:5'VIC-ACCTTGCCAGCTCTGTGCC-3'MGB (SEQ ID NO.126)；
Wherein, 5 ' ends of TaqMan probe sequence are marked with fluorescent reporter group, and 3 ' ends are marked with quenching group.
2. 21 gene detecting kit of breast cancer according to claim 1, which is characterized in that corresponding to detect same gene Primer sets and probe be mixed to form primed probe premixed liquid in proportion, the kit includes respectively corresponding 21 bases of breast cancer 21 kinds of primed probe premixed liquids of cause.
3. 21 gene detecting kit of breast cancer according to claim 2, which is characterized in that corresponding to detect same gene Primer sets concentration dilution to 50-900nM, concentration and probe concentration is mixed to form primed probe premixed liquid after being diluted to 50-500nM.
4. 21 gene detecting kit of breast cancer according to claim 2, which is characterized in that 21 kinds of primed probes are pre- Mixed liquid is attached separately in 21 QPCR reaction tubes.
5. 21 gene detecting kit of breast cancer according to claim 1, which is characterized in that include 2 in the kit × Master Mix solution；2 × Master Mix solution include 10-100mM Tris-HCl, 10-50 mM KCl, 1-10 mM (NH4)2SO4、1.5-6.0mM MgCl2, 0.2-0.5mM dNTP, 0.05-0.2U HotStart TaqDNA polymerization The ddH of enzyme, 10-20% glycerol and surplus2O。
6. the answering in preparation 21 gene detecting kit of breast cancer of primer and probe described in claim 1-5 any one With.
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